The present invention relates to a diagnostic assay method and kit for detecting the presence of a target nucleotide sequence (either DNA or RNA) in a biological sample.
Conventional methods for detecting the presence of a particular polynucleotide in a biological sample typically involve immobilization of nucleic acid of the sample on a surface as the initial step. Once the sample is immobilized, a probe polynucleotide strand, usually tagged with a detectable label such as radioactive phosphorus atoms, is incubated with the immobilized sample so as to bind to the immobilized sample by purine/pyrimidine base sequence-specific complementary base pairing when the immobilized sample contains the target nucleotide sequence. After washing off the labeled probe which has not so hybridized, the presence or absence of label on the support is then determined. Techniques for this determination include exposure of a photographic film, liquid scintillation counting, and fluorescence microscopy. See U.S. Pat. No. 4,358,535 to Falkow et al. (1982).
Ward and coworkers (see EPA No. 63,879 (1982)) have described a variation of this technique in which, rather than tagging the probe directly with a detectable label, the probe is tagged with a nonisotopic substituent such as biotin on certain nucleotides. In such case, after the unhybridized probe is washed off, the support is contacted with a reagent such as avidin linked to an enzyme. The avidin-enzyme complex binds selectively to biotin because of the high avidin-biotin binding affinity, so as to affix enzyme selectively where the target nucleotide sequence has been immobilized on the support. Thereafter, a substrate for the enzyme is added and products of the enzymatic reaction are detected, yielding an amplified signal functionally dependent upon the initial concentration of target nucleotide sequence on the support. See also EPA No. 97,373 of ENZO BIOCHEM, INC (Jan. 4, 1984).
A variation in the above nonisotopic system has also been described in another European patent application of Standard Oil of Illinois (EPA No. 70,687 (1983)) in which, in one form (see pages 8-10 thereof), two nucleic acid probes specific for the target nucleotide sequence are employed. The first probe, which can hybridize to a first portion of the target nucleotide sequence, is affixed to a solid support such that, upon incubation of the solid support with a sample of the biological material, target nucleotide sequences in the sample will bind to the support selectively via this first immobilized probe. Thereafter or concurrently, the second probe, which can hybridize selectively to a second and distinct portion of the target nucleotide sequence, is exposed to the support. Again, if the target nucleotide sequence is present in the biological sample, the second probe will bind selectively to that nucleotide sequence; and a combination structure (or sandwich) will be created linking the second probe to the support via the first probe and the target nucleotide sequence. The published patent application discloses labeling this second probe with a moiety directly or indirectly generating or absorbing specific wavelengths of light (e.g., a fluorescent label, a phosphorescent label or a chemiluminescent label). By separating the support from unbound solution constituents at each stage, the presence of label in the phase with support after the third separation will be a function of the presence and concentration of the target nucleotide sequence in the sample. See also WO No. 83/01459 of Orion-Yhtma Oy (Apr. 29, 1983).
While the above hybridization procedures will detect the presence of target nucleotide sequences in biological samples in many cases, they each have the disadvantage of either multiple steps or steps with necessarily long incubation periods that make them impracticable for easy use in a clinical laboratory. Furthermore, many of these processes suffer from a limited selectivity or sensitivity with regard to interfering polynucleotide sequences or reliable detection of low levels of target nucleotide sequence against the background signal. In particular, nonspecific binding of the labeled probe represents a substantial source of background signal in each process.
Apart from the analysis of biological samples for target nucleotide sequences, various aspects of the physical chemistry of hybridization (formation of double-stranded helices between complementary polynucleotide sequences) have been studied. These studies have included examination of the phenomena of strand migration and displacement in nucleic acid, both in vivo and in vitro. By referring to such studies, however, we do not admit that the phenomena of strand migration and displacement have any obvious applicability to diagnosis and detection. C. Green and C. Tibbetts, Nucleic Acids Research vol. 9, No. 8, pp. 1905-18 (1981), have described the formation of a complex (hybrid) of a 6.1 kb (6100 base long) single-stranded DNA polynucleotide hybridized near its middle (the interval 1.7-3.3 kb) to an end-labeled complementary DNA polynucleotide of 1.6 kb length. Addition to this complex, in solution, of the 6.1 kb complementary strand caused rapid displacement of the labeled polynucleotide (see FIG. 2 on page 1910 of this reference), monitored by taking aliquots of the reaction mixture, separating then by gel chromatography and analyzing then by autoradiography. The displaced 1.6 kb polynucleotide increased steadily from under 10% to over 90% of the radioactive signal in a period of more than 85 minutes (depending upon concentration) with the 1.6/6.1 kb hybrid accounting for the bulk of the remaining radioactivity. The presumed partially displaced intermediate, which would have a total mass equivalent to 13.8 kb of DNA (both long strands and a partially displaced short strand) was apparently not detected. The authors concluded that the initial hybridization of the two 6.1 kb polynucleotides, forming a branched species, was the rate-limiting step; and that displacement along the 1.6 kb paired region of a labeled polynucleotide was very rapid, consistent with a calculated average lifetime of the branched (13.8 kb mass equivalent) species of 0.8 minutes. They indicate the possibility of both single-branched or doubly nucleated (D-looped) intermediate species (illustrated on page 1912 of the reference). In order to better study the phenomenon of branch migration, they attempted to slow the displacement process, by using drugs which might retard the migration phenomenon and/or by using complexes with more than 1.6 kb of hybrid base pairing (see pages 1913-1914 of the reference). It should be noted that the 1.6/6.1.kb species was challenged by Green et al only with the 6.1 kb complementary strand, purified away from any non-specific strands.
A separate issue in nucleic acid biochemistry has been the examination of polymeric species which interact with nucleic acids during strand hydridization processes. Polyethers such as poly(ethylene glycol) have found use in a variety of specific biological or biochemical processes such as viral particle isolation (K. R. Yamamoto et al., Virology 40,734-744 (1970)), nucleic acid purification (B. Alberts in Methods in Enzymology, S. P. Colowick and N. O. Kaplans, eds. (Academic Press, N.Y.), vol. 12, pp. 566-581 (1967)), protein purification (K. G. Ingram, Arch. Biochem. Biophys. 184, 59-68 (1977)), mammalian cell fusion (G. Galfre et al., Nature 266, 550-552 (1977)) and enhancement of specific enzymatic activity (B. H. Pfeiffer and S. B. Zimmerman, Nucleic Acids Research 11,7853-7871 (1983)). M. Renz and C. Kurz, Nucleic Acids Research, vol. 12, 3435-44 (1984) (which may not constitute prior art with respect to the present invention) disclose the use of polyethylene glycol as a volume excluding agent to enhance the rate of hybridization in an immobilized-sample type of DNA probe assay (particularly of the type referred to as a Southern blot). See especially page 3441 of this reference. In this respect other volume exclusion polymers of various types have been shown to enhance the rate of hybridization of matched single strands. See Wetmur et al., Biopolymers, vol. 10, pp. 601-613 (1971) (dextran sulfate), Wahl et al., Proc. Nat. Acad. Sci., vol. 76, pp. 3683 (dextran sulfate). In general, polymers useful in this regard are non-ionic or anionic water-soluble polymers (including polysaccharides) which do not react with DNA. See S. B. Zimmerman et al, Proc. Nat. Acad. Sci., vol. 80, 5852-56 (1983); B. H. Pfeiffer et al, cited above; Water-soluble Synthetic Polymers (P. Molyneux, ed.; CRC Press, Inc., Cleveland, Ohio--two volumes, 1983).